Imaging apparatus associated with an image database

ABSTRACT

The invention concerns an image database ( 30 ) comprising a plurality of tables (TAB) each including fields concerning at least image acquisition conditions, and/or sample identification, the tables being connected to one another in accordance with selected relationships. Each image acquisition comprises a control for displacing the sample-holder, parameters of the filtering means or parameters of the processing means, and an input for values of each acquisition parameter in the corresponding fields of the database tables. The imaging apparatus comprises a consultation unit for interrogating the image database with a request containing at least a criterion corresponding to at least an acquisition parameter, to select images fulfilling said criterion and to display said resulting selected images. The consultation unit comprises processing and analysing means for processing the resulting acquired images, and related data, retrieved or derived from processing said images.

[0001] The present invention relates to the imaging of samples and tothe management of the images with the aid of an integrated andpolyvalent relational database.

[0002] Its general application is to the acquisition and quantitativeanalysis of images and, more particularly, to the applications of thelife sciences based on the high-resolution and high-speed identificationand measurement of DNA (deoxyribonucleic acid).

[0003] One particular application for it is, for example, in studiesinvolving immunofluorescence, cytogenetics, applied genomics, cytology,molecular biology, histo-cytochemistry, pharmacology, toxicology andmolecular pathology etc.

[0004] Numerous apparatuses for imaging biological samples byfluorescence are already known. In the patent FR-A-2532756, for example,there is a description of an apparatus for imaging by fluorescence, inwhich an excitation light beam is directed onto a sample arranged on asample holder. The radiation emitted by the sample thus excited isdirected towards detection means. The signals detected are thenprocessed to acquire a corresponding image of all or some of the sampleobserved. In the case of fluorescence, the excitation beam is filteredin terms of wavelength in order to select the radiation of use forproducing the phenomenon of fluorescence, while the radiation emitted isfiltered in order to stop the radiation of no significance to thephenomenon of fluorescence.

[0005] The prior art also includes imaging apparatuses (in the patentU.S. Pat. No. 4,122,518—CASTLEMAN et al, for example) which furthermorecomprise an electronic control unit capable of adjusting thedisplacement of the sample holder. Generally, the images acquired arestored in digital form in a database. With the aid of such a database,the user is able to perform tasks of data processing, of recognition ofobjects and of their classification (karyotyping). In practice, the userselects an image by its file name, which offers little possibility ofsorting. Moreover, management of the database is generally limited to asingle, relatively static, application with little possibility ofdevelopment, which is awkward in terms of its interactivity with theuser.

[0006] The present invention provides a solution to these problems.

[0007] Thus it aims to provide an integrated polyvalent relational imagedatabase which, when associated with an appropriate imaging apparatus,makes it possible, in particular, to perform selection and processing ofimages according to much richer and more sophisticated criteria thanmerely the name of the file corresponding to the image, in particularaccording to parameters linked to the acquisition of the images and/orto the identification of the sample and/or to the data linked to and/orderived from and/or extracted from these images.

[0008] It thus relates to an imaging apparatus of the type comprising:

[0009] a sample holder that can be adjusted at least in the X and Ydirections;

[0010] an illumination device comprising a light source capable ofgenerating an illumination beam;

[0011] first transfer means capable of directing the said illuminationbeam onto a sample arranged on the sample holder;

[0012] a receiving device comprising second transfer means capable ofdirecting the radiation emitted and/or transmitted by the sample thusilluminated to detection means mounted at the output of the receivingdevice, the first and/or second transfer means comprising adjustablefiltering means;

[0013] processing means for processing the signals thus detected by thedetection means in order to acquire a corresponding image of at leastone zone of the sample;

[0014] an electronic control unit capable of controlling at least thedisplacement of the sample holder, the parameters of the filtering meansand/or the parameters of the processing means according to a pluralityof modes of image acquisition; and

[0015] an image database linked to the processing means and capable ofcontaining the images thus acquired.

[0016] According to a general definition of the invention, the imagedatabase comprises a plurality of tables, each comprising fieldsrelating at least to the conditions of acquisition of the images and/orto the identification of the sample, the tables being linked to eachother according to selected relationships;

[0017] each image acquisition comprising a command for the displacementof the sample holder and/or parameters of the filtering means and/orparameters of the processing means and capture of the values of eachacquisition parameter in the corresponding fields of the tables of thedatabase;

[0018] the imaging apparatus furthermore comprising a look-up unit forinterrogating the image database in accordance with at least one querycontaining at least one criterion corresponding to at least oneacquisition parameter so as to select the images that meet the saidcriterion and display and/or process the said images thus selected.

[0019] The applicant has thus observed in a surprising manner that inmastering and controlling the adjustment especially of the displacementparameters of the sample holder, the parameters of the filtering meansand/or the parameters of the processing means, they have at theirdisposal numerous items of information linked to the acquisition of theimages, the values of which can be captured and interlinked in therespective tables of the image database, making it possible to provide arelational, integrated and polyvalent image database that is capable ofbeing interrogated in accordance with queries containing criteriacorresponding at least to the conditions of acquisition of the imagesand/or to the identification of the sample. The result is that the userof such an imaging apparatus according to the invention has at hisdisposal an image database with multiple tables capable of beinginterrogated in accordance with rich, sophisticated and dynamic querieslinked directly at least to the conditions of acquisition of the imagesand/or to the identification of the sample, something that prior-artdatabases do not provide.

[0020] According to another significant characteristic of the invention,the look-up unit furthermore comprises means of processing and/oranalysis capable of processing and/or analysing the images thus acquiredas well as the data linked to and/or extracted from and/or derived fromthe processing and/or analysis of these images.

[0021] Finally, the database is polyvalent since it is capable of beingused for any imaging application (fluorescence, transmission microscopy,bright field etc.), which is not the case with prior-art databases,which are generally dedicated to a single application.

[0022] In practice, the fields of the multiple tables belong to thegroup formed by the X, Y and/or Z coordinates of the sample holder, theconditions of operation of the light source; the wavelength, bandwidth,attenuation factor, and/or position of the filtering means; the timeconditions of image acquisition; the nature and operating parameters ofthe elements of the first and second transfer means; the operatingparameters of the detection means; and the operating parameters of theprocessing means.

[0023] The look-up unit preferably comprises image-processing means ofthe type comprising contrast, linear filtering, operations ofmathematical morphology and segmentation, as well as means ofcalculation of the type comprising characterisation of shape, density,texture, topology and spatial organisation and means of presentingresults tables of the type comprising spreadsheets or tools forstatistical analysis and interpretation.

[0024] Other characteristics and advantages of the invention will becomeapparent in light of the detailed description below and of the drawings,in which:

[0025]FIG. 1 shows schematically an imaging apparatus according to theinvention;

[0026]FIG. 2 is an organigram illustrating the acquisition of the imagesand the capture of the acquisition values in the image databaseaccording to the invention;

[0027]FIG. 3 shows a diagram of the image database according to theinvention representing the relationships between the multiple tables;

[0028]FIG. 4 shows an organigram illustrating the selection of an imagestored in the image database according to the invention; and

[0029]FIG. 5 shows an organigram illustrating an example of processingand analysis of images stored in the image database according to theinvention.

[0030] Referring to FIG. 1, this shows an apparatus for imaging byfluorescence, which is associated with an image database according tothe invention. It is obvious that the image database according to theinvention can be associated with any other type of imaging apparatus,e.g. an optical microscope (transmission, bright field), but also ascanning electron microscope, confocal microscope etc.

[0031] The imaging apparatus in FIG. 1 comprises a sample holder 2designed to support a sample 4. The sample holder 2 can be moved in atleast two directions perpendicular to the plane of the sample (X and Ydirections). The sample holder 2 is supported by a stand 6. The sampleholder 2 is likewise adjustable in height (direction Z or of focus). Thesample holder is, for example, a table driven by two motors, one alongthe X axis and the other along the Y axis. A control unit 8 controls thedisplacement of the motor-driven table 2 by sending selected commands 9and by receiving responses 11 to the said commands 9.

[0032] A light source 10 is capable of generating an excitation beam 12(for fluorescence) or an illumination beam (for observation bytransmission). The excitation beam 12 is directed onto the sample 4 viatransfer means that can include a specific optical separation device 14(dichroic mirror) and an optical magnification device 16 (objective).The dichroic mirror 14 makes it possible to send the excitationradiation 12 towards the sample 4 and to allow the fluorescence emission22 to pass towards the eye of the observer.

[0033] As a general rule, the fluorescence objective 16 magnifies by 10to 100 times.

[0034] The light source 10 is, for example, a mercury vapour arc lamp, axenon arc lamp or a halogen lamp (for transmission microscopy).

[0035] Excitation filters 18 arranged downstream of the source ofradiation 10 make it possible to select the radiation that is useful forproducing fluorescence.

[0036] Stop filters 20 arranged between the sample 4 and the eyeeliminate the radiation that is re-emitted by the sample 4 and is of nosignificance to the phenomenon of fluorescence.

[0037] Owing to the fact that the maxima of the wavelengths of theexcitation radiation and of the emission of fluorescence are close toeach other (of the order of around ten nanometres), the choice ofexcitation filters and stop filters is an extremely delicate matter inpractice. The optical means of observation are generally formed by amicroscope specially adapted to the technique of observation by means offluorescence.

[0038] The sample holder 2 is capable of carrying a plurality ofsamples. The electronic control unit 8 is capable of displacing the saidsample holder under one or more magnifying objectives 16 of variouskinds and an eyepiece. The objective or objectives 16 provides orprovide an actual image of the object observed, while the eyepieceprovides the eye with an enlarged virtual image.

[0039] In practice, the excitation filters 18 and stop filters 20 arecarried by rotatable wheels. An electronic control unit 24 sendscommands 19 and 21 towards the excitation filters 18 and the stopfilters 20 in order to select the corresponding filter at the discretionof the user.

[0040] The electronic control unit 24 likewise sends commands 23 to thelight source according to selected parameters, in particular concerningthe intensity of the lamp and the adjustment of the diaphragm (whererequired).

[0041] The electronic control units 8 and 24 are driven according toprograms contained in a microcomputer 26, the operating system of whichis, for example, WINDOWS NT (registered trademark).

[0042] The fluorescent emission radiation 22 is detected by a camera 28with a network of CCD-type semiconductor detectors. The signals detectedby the camera 28 are recorded in digital memories 29 for the purpose ofprocessing by image-processing programs and are stored in magneticmemories (not shown) in the microcomputer 26. The digital memories canbe arranged on dedicated cards or in the microcomputer. The images arestored in the TIFF format (Tagged image file format), for example.

[0043] The camera 28 is preferably a high-resolution camera, 1280×1024pixels for example, with a depth of measurement of the signal of atleast 10 bits (1024 levels) per pixel, and with cooling by the Peltiereffect. The sensor is of the ⅔″ interline type. The rate of acquisitionis 7 images per second at high resolution, for example. The camera 28has an interface card of the PCI bus type linked to the microcomputer26.

[0044] The parameters of the camera, in particular gain, offset and timeof exposure can be adjusted electronically. The camera is capable oftaking a single image or a series of images. After acquisition, theimages are automatically placed in the “image” table of the database,which will be described in greater detail below.

[0045] According to the invention, the microcomputer 26 is capable ofmanaging an image database 30 that not only stores the images acquiredin this way but also, above all, all the parameters relating to theconditions in which these images were acquired and subsequently all thevalues of the parameters and information obtained from the processingand analysis of these images. The microcomputer 26 includes a look-upunit 32 which makes it possible to interrogate the database 30.

[0046] Referring to FIG. 2, an organigram illustrating in a generalmanner the acquisition of an image with the imaging apparatus accordingto the invention has been shown.

[0047] As described above, acquisition of an image is preceded by thecontrol (step 1) of the elements participating in the acquisition of theimage. Control is concerned particularly with the adjustment of thesample holder, the filtering means, the camera, the processing meansand/or the quantitative analysis means.

[0048] After acquisition of the image, provision is made to capture thevalues of the parameters linked to the acquisition in the tables of thedatabase which will be described in greater detail below (step 2). Theword “capture” is here intended to mean the automatic operation ofrecording the values of the parameters or manual capture of these valuesby an operator.

[0049] The image file containing the image thus acquired is likewisestored (step 3).

[0050] In practice, the displacement of the sample holder in the X and Ydirections in certain applications, such as that referred to as “captureby 2-D scanning”, allows the successive examination of a plurality ofcells containing a preparation to be observed or the whole of aparticular structure constituting the sample.

[0051] In other applications, particularly those involving fluorescence,it is necessary to perform multispectral capture. In this case, thecamera examines the sample successively through the optical observationmeans and through at least two different coloured or selective filterschosen as a function of the wavelength of the light radiation that theyallow to pass, either for transmission through the sample or for theradiation due to fluorescence.

[0052] The storage means 29 record the electrical signals correspondingto the individual coloured images viewed and recorded digitally by thecamera. Means simultaneously feed back the electrical signals to theoutput of the said memories, and means vary the gain and exposure timeof the camera 28 in such a way that the amplitude of the output signalsof the latter is always greater than a predetermined threshold and takesaccount of nonrepresentative signals (noise), while at the same timecorrecting them.

[0053] In the case of immunofluorescence, it is generally sufficient touse only two different coloured images, namely a red image and a greenimage. In certain cases, however, it may be advantageous to use a thirdimage, a blue image for example.

[0054] It should be pointed out that the filter-holder wheels eachcomprise a disc provided with 8 coloured filters set in rotation in sucha way as to move the filters successively into a position perpendicularto the light beam. A disc of this kind is driven by a motor, of thestepping type for example, the operation of which is, of course,synchronised with the rest of the apparatus according to the inventionand, in particular, the storage of the signals corresponding to theindividual coloured images. To ensure such synchronisation at least inpart, it is advantageous to provide fixed sensors, optoelectronic and/ormagnetic sensors, for example, co-operating with markers on the saiddisc.

[0055] The imaging apparatus according to the invention is capable ofautomatically acquiring images according to several modes of acquisitionor acquisition protocols.

[0056] One of the protocols consists in automatically acquiring a seriesof images of the same sample. The sample holder is displaced accordingto selected movements in the X and Y directions. This protocol isreferred to as “capture by 2-D scanning”. More precisely, it is possiblewith this protocol to scan the whole of a sample systematically at ahigh magnification (e.g. ×40 or ×1000) along the X and Y axes.Observation can also be performed within a region of a sample, theposition and surface area of which are defined graphically by the useras the result of a dialogue with the processing means, or arepre-programmed with the aid of an algorithm that makes use of a prioriknowledge of the spatial structure of the sample.

[0057] Another protocol can include monitoring of the displacementsalong the Z axis. This protocol thus consists in acquiring severalplanes or sections of the same sample (batch on the Z axis). Thisprotocol is referred to as “multi-layer 3-D capture”. More precisely, itis possible with this protocol to acquire a batch of several sections ofa biological sample of a certain thickness, the same area or part of asample being observed and captured in the different planes or differentpositions of the sample carrier in the Z direction (expressed to aprecision of a hundredth of a micron), corresponding to positioning atthe focal point, this being performed by software. This function is ofparticular significance for the identification and measurement ofobjects (DNA probe) of very small size situated at different levelswithin the thickness of a sample in different conditions of light forobservation.

[0058] Yet another protocol consists in acquiring a set of images of thesame area or part of the sample using different excitation lights orfluorescent lights or different visualisation colours (red, green, blue)corresponding to the different excitation and stop filters. Thisprotocol is referred to as “multispectral capture”.

[0059] Yet another protocol consists in acquiring images according todifferent acquisition configurations or modalities, e.g. fluorescence,bright field, transmission). This mode is referred to as multimodal. Atthe end of this mode, the user can merge the resulting images forprocessing and subsequent interpretation.

[0060] It is also possible to monitor the time over which the images aretaken and the adjustments of the capture electronics. For example, atime sequence of images, that is to say images acquired over a specifiedtotal time, is acquired at a parameterisable interval.

[0061] Moreover, the user can combine several protocols depending on theapplication envisaged. These acquisition protocols can furthermore besupplemented by the automatic adjustment, in transmission and/orfluorescent mode, of the positioning of a shutter to protect the sample,and by the processing of images online (during capture).

[0062] All the images thus acquired can be stored in the image databasewhich will be described in greater detail below. These images stored inthis way are advantageously capable of being processed, reloaded andmerged to form subsets of images.

[0063] It is possible, for example, to implement various functions in acompletely automatic way during programmed acquisition protocols. Theuser can also create protocols that allow combination of the functionsdescribed above. For example, the user can select automatic modescomprising time sequences and multispectral series so as toautomatically perform measurements of the variations over time of theconcentrations of ions, such as calcium, hydrogen, sodium, chlorine etc.within cells, especially when they are excited by drugs or by electricalsignals.

[0064] In the same way, it is possible, by selecting the modescomprising a multi-plane series on the Z axis and scanning regions onthe X and Y axes, to automatically carry out imaging studies by volume(three-dimensional) at very high spatial definition. Combination ofthese modes with the multispectral mode makes it possible to add thespectral dimension to the series of images obtained automatically(perfecting of processes for the development of medicaments or qualitycontrol in the production of industrial biological solutions).

[0065] Referring to FIG. 3, the image database according to theinvention comprises a plurality of tables of parameters TAB, identifiedindividually as TAB1 to TAB12. Each table comprises a list of parametersor fields relating at least to the displacement of the sample holder, tothe filtering, excitation and stop means, to the means of processing thesignals detected by the CCD camera and/or to the identification of thesample.

[0066] Table TAB1, “preparations”, relates to the preparations linked tothe application.

[0067] In practice, table TAB1, “preparations”, comprises four fieldsCH, identified individually as CH1 to CH4. Field CH1 relates to the nameof the preparation. Field CH2 relates to the date of preparation. FieldCH3 relates to the nature of the preparation. Field CH4 is intended toreceive a description of the preparation.

[0068] Table TAB2, “slides”, relates to the slides.

[0069] In practice, table TAB2 comprises five fields. The first fieldrelates to the name of the slide. The second field relates to the typeof structure of the slide, e.g. a matrix of 8×3 cells. The third fieldrelates to the region or zone of the sample to be observed. The fourthfield relates to the position of the slide to be analysed in the list ofX, Y and Z positions of the said slide. The fifth field relates to thefocussing marks.

[0070] Table TAB3, “cameras”, relates to the means of detection. Thistable comprises six fields. The first field relates to the name of thecamera. The second field relates to the channel by which the camera isconnected to the microcomputer in order to ensure capture of the images.The three [sic] to sixth fields relate to the geometry (size, number ofpixels etc.) of the CCD matrix of the camera.

[0071] Table TAB4, “acquisition status”, relates to the acquisitionstatus of the images. It comprises ten fields. The first field relatesto the identification of the acquisition field. The second fieldcorresponds to the parameter relating to the intensity of the lamp. Thethird field corresponds to the aperture of the diaphragm of the lamp.The fourth and fifth fields relate to the negative and positivereferences of the channel for connecting the camera to themicrocomputer. The seventh field relates to the gain of the camera. Theeighth field relates to the time of exposure of the camera. The ninthfield relates to the number of layers in the Z direction which are to beacquired, merged and/or processed. Finally, the tenth field relates tothe distance between the different layers, the distance being expressedin micrometres.

[0072] Table TAB5, “scenes”, relates to the scenes. This table comprisessixteen fields.

[0073] The first field comprises the unique identification number of thescene. The second field relates to the name of the preparation. Thisfield is linked to the “preparation” field of table TAB1. Therelationship between table TAB1 and TAB5 is of the one-to-several type,that is to say that one preparation can have several scenes. The thirdfield relates to the name of the slide. This field is linked to the“slide” field of table TAB2. The relationship between TAB2 and TAB5 isof the one-to-several type. The fourth field is intended for comments.The fifth, sixth, seventh and eighth fields are intended for theposition of the scene in the mosaic representing the scenes as a whole.If the mosaic of scenes is not referenced using the metric system, theninth and tenth fields relate to the numbers, expressed in rows andcolumns, by which the scene is referenced in the mosaic. The eleventhand twelfth fields are intended for the aspect ratio and the calibrationof images. This aspect ratio can correspond to the height of the pixeldivided by its width. For images in the metric system, the width of thepixel is indicated in micrometres. Finally, the four last fields relateto absolute positions and relative positions on the X and Y axes.

[0074] Table TAB6, “modes”, relates to the modes of acquisition. Itcomprises 15 fields.

[0075] The first field relates to the name of the mode of acquisition.

[0076] The second field relates to the type of imaging by whichobservation of the sample is carried out (fluorescence, transmission,bright field).

[0077] The third field relates to the objective. This field is linked totable TAB8, which will be described below. The relationship between TAB6and TAB8 is of the several-to-one type.

[0078] The four [sic] to seventh fields relate to the name of thefilters linked to the table relating to the filters, TAB9, which will bedescribed in greater detail below. The filter fields are linked byseveral-to-one relationships.

[0079] The eighth field relates to the name of the camera. This field islinked to table TAB3 according to a relationship of the several-to-onetype between TAB6 and TAB3.

[0080] The ninth field corresponds to the chronological order ofacquisition (e.g. blue filter before red filter).

[0081] The tenth field is a field reserved for comments.

[0082] The coefficients which allow balancing between the colours (red,green and blue) are grouped in the eleventh field.

[0083] The exposure limits of the camera are expressed in the twelfthand thirteenth fields: low-level exposure, high-level exposure.

[0084] A correction factor on the Z axis to correct the shifts due tochromatic aberrations and focussing is provided in the fourteenth field.

[0085] Finally, a field called “acquisition” is provided. This field islinked to table TAB4 and the corresponding field by a one-to-onerelationship.

[0086] Table TAB7, “images”, relates to the images thus acquiredaccording to the invention. It comprises twelve fields, for example.

[0087] The first field is the alphanumeric identification number of theimage. This field corresponds to the name of the image file.

[0088] Field number two corresponds to the identification number of thescene. This field is linked to the scene field of table TAB5 accordingto a several-to-one relationship of TAB7 to TAB5.

[0089] Field number 3 corresponds to the name of the mode. This field islinked to the corresponding mode field of table TAB6 according to aseveral-to-one relationship of TAB7 to TAB6.

[0090] An identification number of the parent image is provided for thechild images (field No. 4) derived from a selected processing operationon the parent image or images.

[0091] An “event” field (field No. 5) is intended to receive theinformation recorded during the acquisition of the images.

[0092] The Z coordinates are stored in the “Z” field (field No. 6). Theduration of acquisition or relative time of image capture in seconds isto be stored in field No. 7. The colour balance coefficients (red, greenand blue) are stored in field No. 8.

[0093] The state of the objects defined in the images (masks, labels)are [sic] identified in the status field (field No. 9). Thequantification field is provided (field No. 10). The date of creation oracquisition of images is likewise provided (field No. 11), as is a finalfield (field No. 12) relating to the objects in the image (masks, labelsetc.).

[0094] Table TAB8 relates to the objectives used by the imagingapparatus. It comprises eight fields. Field No. 1 corresponds to thename of the objective. Field No. 2 relates to the magnification factor.Field No. 3 relates to the numerical aperture of the objective. Thedepth of the focal plane is indicated in field No. 4. The position ofthe objective in the turret is given in field No. 5. The X and Y offsetrelative to the optical axis is provided in fields No. 6 and No. 7.Finally, field No. 8 relates to the type of objective (dry, oil, water).

[0095] Table TAB9 corresponds to the filter table. This table containsthe name of the filter, the centre of the transmission band, the widthof the transmission band in nanometres and the position of the filter onthe wheel. The attenuation factor is also indicated with a factor equalto one for total transparency and a factor equal to zero for completeblocking.

[0096] Table TAB12, “image acquisition”, is substantially identical totable TAB4. It furthermore comprises the “image” field (field No. 1),which is linked according to a one-to-one relationship to the “image”field of table TAB7. Table TAB12 also describes the electronic andmechanical adjustments of the various devices participating in theacquisition of the image at the moment of the said acquisition. Thistable is useful since it contains the main fields relating to theconditions of acquisition of the images. With the aid of this table, theuser can easily select and display an image according to the criteria ofacquisition.

[0097] Tables TAB10 and TAB11 relate to the results. For each image, aset of objects or zones of interest (e.g. cells, chromosomes orparticles) is distinguished.

[0098] Table TAB10 is called “results-objects”. It contains threefields. The first field relates to the objects in the image. The secondfield relates to the “image” field. This field is linked to the “image”field of table TAB7 according to a several-to-one relationship of TAB10to TAB7. The third field and the following fields relate to the specificparameters (surface area, perimeter, intensity of fluorescence) of eachobject.

[0099] On each object, it is furthermore possible to observe zones ofinterest or spots of even smaller dimensions than the said objects.Table TAB11, which is called “results-spots”, serves precisely tocontain information on these zones of interest.

[0100] Table TAB11 comprises four fields. The “spot” field relates to azone of interest on an object (e.g. silver grain in hybridisation insitu, fluorescent point). The “image” field is linked to the “image”field of table TAB7 according to a one-to-one relationship. The thirdfield relates to the objects. This field is linked is linked [sic] tothe “objects” field of table TAB10 according to a one-to-severalrelationship of TAB10 to TAB11. Finally, the final fields relate to thespecific parameters of the spot.

[0101] Here, the innovation consists, in particular, in the fact ofhaving several tables, each containing fields relating to theacquisition of the images, the identification of the sample and theprocessing and analysis of the data linked to and/or derived from and/orextracted from these images.

[0102] Referring to FIG. 4, it is possible to interrogate the database30 with queries containing criteria corresponding to at least oneacquisition parameter (step 10), to select the images that meet the saidcriteria (step 11) and display and/or process the said images thusselected (step 12).

[0103] In general terms, a query allows specific data to be sought,sorted and retrieved. The query selects a set of recordings that meetthe criteria defined by the user. The result of the query forms adynamic answer sheet.

[0104] For example, the query can comprise criteria relating to theidentification of the sample (in this case with the fields of the tablesentitled “preparations”, “slides” and “scenes”), and to the conditionsof acquisition of the images (in this case with the fields of the tablesentitled “acquisition status”, “modes”, “objectives”, “filters” and“image acquisition”. Selection can also be performed with the aid of the“images” table.

[0105] Of course, the database 30 can be updated with the aid of forms.These forms also enable the data in the multiple tables to be consultedand supplemented with formatting that can be personalised.

[0106] Output states allow the information to be presented in writtenform or as a display on the access terminal.

[0107] The query may also require special processing of the raw imagesthus acquired. For example, the query may consist in measuring anddisplaying the level of intensity of green fluorescence within objectsas identified in an image obtained with a blue filter, for example. Inresponse to this query, the look-up unit interrogates the database,which then finds the corresponding green image thanks to the imageacquisition parameters (image, scene, X and Y positions, filter, camera,exposure time etc.).

[0108] The appropriate image processing (in this case calculation of thelevel of intensity) is applied to the objects in the green image thusselected. The result of the calculation is output to the look-up unit.

[0109] The results table can also be interactive. Thus, it is possible,with a click of a computer mouse on a chosen value in a field of thetable, for the user to highlight a chosen region of the image andclicking on an event in the image allows the user to highlight thecorresponding entry (or field) in the table.

[0110] Conversely, the parameters (or fields) of tables TAB10, TAB11 andTAB12 as well as those of the “images” table, TAB7, (especially thefields “X”, “Y”, “Z”, “mode of acquisition”, “time of acquisition” etc.)make it possible to find the image or images corresponding to theseparameters in the database and to display them.

[0111] It should be mentioned that the structure of the databaseaccording to the invention makes it possible to improve the analysis ofthe images in terms of flexibility of use and richness of processingfunctions.

[0112] The database according to the invention can be used, for example,in an imaging procedure intended to detect and measure theamplifications (number of copies) and deletions (suppressions) of DNAsequences in chromosomes of tumour (cancerous) cells in comparison withthe same chromosomes of normal cells. This procedure, referred to as“CGH” for “Genome Comparison after Hybridisation”, is a test thatprovides unique information as an aid to interpreting and diagnosingcancerous tumours.

[0113] The display of the profiles (level of intensity of fluorescence)can also be interactive. Thus a user can highlight selected pixels in animage. It is likewise possible for images derived from a particularprocessing operation (e.g. normalisation) also to be added to thedatabase. These images can advantageously be displayed using anappropriate range of colours to make them easier to interpret.Calculations or thresholding can likewise be performed on the imageswith the aid of a spreadsheet or some other tool for statisticalanalysis and interpretation.

[0114] One specific application for the automatic procedure of analysingthe data and the use of the database according to the invention is inhigh-magnification fluorescence microscopy. In this application, theacquisition protocol entails scanning systematically along the X and Yaxes, autofocussing for each sample region observed and automaticmulti-colour operation, in the present case of three selective filtersfor excitation light and blue, red and green emission.

[0115] For example (FIG. 5), the images obtained in blue mode (step 20)contain information or represent the chromosomes present in each of thecells observed. A first, analysis algorithm, capable of combining theshape of the objects and the density of their blue colouration, makes itpossible to detect the contours of these objects. At the output of thiscontour detection step, a binary image (containing zeros or ones at eachpoint) is generated, this being referred to as an object mask (theobjects being chromosomes in this case).

[0116] A second, recognition algorithm (or shape characterisationalgorithm) makes it possible (step 21), for example, to locate preciselythe “centromeres” of each chromosome detected and to classify them bycomparison with a reference database leading to the generation of animage of a particular type referred to as a karyotype, in which all thechromosomes of the cell observed are shown according to their pairingand their membership of a conventionally defined class in the referencedatabase.

[0117] In practice, the green and red images obtained for each of thecells observed are automatically normalised by a method which itselfmakes use of the integrated structure of the database. The backgroundlevel is evaluated by two alternative techniques, either as the value ofthe mode of the histogram for the distribution of intensity in the imageon the outside of the contours of the chromosomes as detected by thefirst analysis algorithm, or as the value calculated in the immediateproximity (vicinity) downstream of a digital filter of the “top hat”type. These two values for the green background and the red backgroundare subtracted from the green and the red image respectively. Finally,the value of the normalised ratio of the intensities measured iscalculated point by point for each cell observed and on the inside ofthe contour of each chromosome.

[0118] During these steps 20 and 21, numerous queries are input into theimage database according to the invention. These queries are formulatedusing the fields of the tables TAB described with reference to FIG. 3.

[0119] For example, selection by means of the “image”, “scene” and“mode” fields of table TAB7 and of the “filter 1” field of table TAB6makes it possible to identify, select and display an image of a zone ofa sample obtained in blue mode. Selection of the “filter 2” field oftable TAB6 makes it possible to identify, select and display the imageof the same scene obtained in red mode. Likewise, selection of the“filter 3” field of table TAB6 makes it possible to identify, select anddisplay the image of the same scene obtained in green mode.

[0120] The structure of the database furthermore enables the whole ofthe automatic procedure not only to be made possible but also to be madeparticularly efficient. In particular, the fact of rapidly finding theblue-coloured image for each cell observed with the mask (binary image)of certain objects of interest (chromosomes) and all the imagesassociated with this cell (or more generally scenes or parts of asample) that has been observed, in this case the green and red images,representative respectively of the marking of normal and tumorous DNA.The image of the relationship between the preceding images is itselflikewise integrated into the database of this specific study. This imageof the relationship between the preceding images can be analysed easily,is capable of being archived, referenced and transmitted etc. with theaid of the tools for managing this image database.

[0121] For example, the axes of the chromosomes can be calculatedautomatically and used in a function for generating and representingprofiles of the intensity of fluorescence for each chromosome observedin green and red light. The structure of these objects in the databaseallows a unique function for representing visual links on the same workscreen between the position and the value on a chromosome profile andthe position on the image of the chromosome itself.

[0122] After their normalisation and representation according to acolour code adapted to interpretation of the values of therelationships, the images obtained for each cell or part of a sampleobserved can furthermore be subjected to algorithms for detecting aregion of localisation of the amplifications and deletions.

[0123] At the end of this automatic detection, a result table iscreated, integrated with the database and displayed (step 22). For eachof the regions, this table contains the class of the chromosome on whichit is situated, its precise position defined with respect to thecentromere and to the length of the chromosome, its surface area, thestatistically significant values for the intensity of the green and redcolour and for the ratio of these values, for example.

[0124] The results table (step 23) is itself represented on the screenof the access console by graphical dialogue functions that allowinteractivity (made very easy by the use of the mouse and/or of thekeyboard) between the various fields of the tables of the image database(image, object detected by analysis of these images, corresponding valuein rows and columns of the spreadsheet). Statistical analysis andinterpretation tools enable these results tables to be analysedefficiently and interactively.

1. Imaging apparatus of the type comprising: a sample holder (2) thatcan be adjusted at least in the X and Y directions; an illuminationdevice comprising a light source (10) capable of generating anillumination beam (12), first transfer means (14, 16) capable ofdirecting the said illumination beam (12) onto a sample (4) arranged onthe sample holder (2); a receiving device comprising second transfermeans (16, 14) capable of directing the radiation (22) emitted and/ortransmitted by the sample thus illuminated to detection means (28)mounted at the output of the receiving device, the first and/or secondtransfer means comprising adjustable filtering means (18, 20);processing means (26) for processing the signals thus detected by thedetection means in order to acquire a corresponding image of at leastone zone of the sample; an electronic control unit (24, 8) capable ofcontrolling at least the adjustment of the sample holder and/or theparameters of the filtering means (18, 20) and/or the parameters of theprocessing means (26) according to a plurality of modes of imageacquisition, an image database (30) linked to the processing means andcapable of containing the images thus acquired, characterised in thatthe image database (30) comprises a plurality of tables (TAB), eachcomprising fields relating at least to the conditions of acquisition ofthe images and/or to the identification of the sample, the tables beinglinked to each other according to selected relationships; in that eachimage acquisition comprises a command for the displacement of the sampleholder and/or parameters of the filtering means and/or parameters of theprocessing means and capture of the values of each acquisition parameterin the corresponding fields of the tables of the database; and in thatthe imaging apparatus furthermore comprising [sic] a look-up unit forinterrogating the image database in accordance with at least one querycontaining at least one criterion corresponding to at least oneacquisition parameter so as to select the images that meet the saidcriterion and display and/or process the said images thus selected. 2.Apparatus according to claim 1, characterised in that the look-up unitfurthermore comprises means of processing and analysis capable ofprocessing and/or analysing the images thus acquired as well as the datalinked to and/or extracted and/or derived from the processing and/oranalysis of these images.
 3. Apparatus according to claim 2,characterised in that the image-processing means of the look-up unit areof the type comprising contrast, linear filtering, operations ofmathematical morphology, segmentation, as well as means of calculationof the type comprising characterisation of shape, density, texture,topology and spatial organisation and means of presenting results tablesof the type comprising spreadsheets or tools for statistical analysisand interpretation.
 4. Apparatus according to claim 1, characterised inthat the sample holder (2) is furthermore adjustable in the Z direction.5. Apparatus according to claim 1 or claim 4, characterised in that theparameters relating to the conditions of image acquisition belong to thegroup formed by the X, Y or Z coordinates of the sample holder (2); theconditions of operation of the light source (10); the wavelength,bandwidth, attenuation factor, position of the filtering means (18 and20); the time conditions of the acquisition of the images; the natureand operating parameters of the elements forming the first and secondtransfer means (14, 16); the operating parameters of the detection means(28) and the operating parameters of the processing means (26). 6.Apparatus according to one of the preceding claims, characterised inthat at least certain tables are interlinked according to relationshipsof the one-to-several, one-to-one and/or several-to-one type. 7.Apparatus according to one of the preceding claims, characterised inthat the database is of the relational, integrated and polyvalent type.